Many chemical and biochemical assays employ an interactive material which is retained upon a support member. In the context of this disclosure, an interactive material is meant to include a material which, in at least one step of the assay, interacts with other chemical components of the assay. The interaction can comprise a binding reaction, or a reaction which generates new chemical species or which alters the configuration of a chemical species. The interactive materials can comprise molecular materials, macro molecular materials, or assemblages of species such as membranes, layered structures and other molecules that interact with chemical components of the assay. For example, the interactive material can include antibodies, antigens, enzymes or other catalytic species, substrates for the enzymes, reducing agents, oxidizing agents, proteins, nucleic acids, lipid structures, receptors, membranes, carbohydrates and various polymeric materials.
Frequently, at least one interactive material in a chemical reaction is retained upon a support member which may comprise a plate, a body of beads or other particulate material, tubes, rings, a porous matrix or other material of various designs. Within the context of this disclosure, any such support structure will be referred to as a support body. One particularly important class of support bodies comprise those bodies having a scintillator material incorporated thereinto. As is known in the art, a scintillator material generates light in response to energy input, most typically from a particle or high energy photon generated in a radiochemical decay process. Scintillator coated supports are frequently used in combination with radio chemical agents for a variety of assay procedures wherein light generated by the scintillator support, is indicative of the presence or amount of specific materials.
In general, the prior art has implemented a variety of approaches towards retaining interacting materials on support bodies. Any such methods must adequately secure the interactive material to this support so that it will be retained thereupon throughout storage and use of the coated support as is necessary. In the prior art, interactive materials have been retained on a support, by hydrophilic/hydrophobic interactions, or physical processes such as adsorption and the like. In some instances, a support body is coated with a material which facilitates adhesion of the interactive material. In some instances, materials have been bonded to supports through covalent binding; however, such prior art techniques required pretreatment of the support, or the interactive material, under fairly extreme conditions in order to provide chemically modified sites through which covalent bonding is accomplished. For example, .sup.60 Co irradiation has been employed to graft carboxyl groups onto a polymeric assay plate in order to modify the plate so that proteins can be subsequently affixed thereto by EDAC activated covalent binding. (Larssen et al. 1987). Such surface modification steps are costly and difficult to implement. Therefore, there is a need for the methods of the present invention, which permit interactive materials to be covalently retained upon supports without the need for irradiating, grafting, chemically modifying or otherwise pretreating the support or the interactive material. Any materials or methods used to affix the interactive material to a support body must not interfere with the intended use of the interactive material or the resultant combination. This presents a significant problem when scintillator support bodies are employed, since a number of materials can quench scintillation or otherwise interfere with the use of the substrate.
As will be explained in greater detail herein below, the present invention is directed to methods and materials wherein an interactive material is retained on a support body by covalent interaction with, or initiated by, a linker material. The linker material is selected to covalently bind to the interactive material and to also be capable of binding, by covalent binding or otherwise, to the support body. Alternatively, the linker material will cause the interactive material to covalently bind to the support, while it, itself, is not incorporated in the final bond. In such instances, the linker material is akin to a catalyst. Within the context of this disclosure, the interactive material is still considered to be covalently bonded to the support by the linker, since the linker causes and participates in the binding. Covalent binding is a chemical binding wherein a bond is created by the sharing of electrons between atoms, and as such, is distinguished from physical processes such as absorption, and from other types of chemical bonds such as purely ionic bonds. It is to be understood that, in accord with chemical bonding theory, covalent bonds can have some degree of ionic character and still be considered covalent bonds; accordingly, within the context of this disclosure, covalent bonds are to be understood to be bonds in which there is some degree of electron sharing, even though there may be some degree of ionic character in that bond.
The methods and materials of the present invention provide for the efficient attachment of a variety of interactive materials to various substrates. It is significant that the attachment materials and methods of the present invention may be employed in those instances where scintillator support bodies are utilized, and will not adversely affect the use of such supports. The materials and methods of the present invention may be employed in connection with a variety of applications wherein interactive materials are retained upon support bodies, and one application comprises assays. One type of assay in which the present invention has particular utility is an enzyme assay wherein a substrate for the enzyme is immobilized upon a support body. One specific type of such assay utilizes a synthetic membrane as a substrate, and the present invention is readily employed to attach such synthetic membrane materials to a variety of support bodies including scintillator support bodies.
Because many intracellular and intercellular processes are membrane mediated, there has been a great deal of research on the reconstitution of biological membranes as a method to study these processes. Since the original development of a procedure to form artificial planar phospholipid bilayer membranes (Mueller et al., 1962) and the demonstration of the fusion of vesicles that contain ion channels to planar membranes (Miller and Racker, 1976), studies have employed a variety of artificial membrane systems and various methods for studying functional molecules that are incorporated in or bound to biological membranes (Hoekstra and Duzgunes, 1993). Some researchers have incorporated biotinylated lipids (e.g., see Wright and Huang, 1992) or functional enzymes (e.g., see Hianik et al., 1996) into artificial membranes, but the purpose has been to study properties of the membranes or the incorporated molecules, not to use the synthetic membranes as a tool to study the properties of exogenous molecules as in the present invention. For a review of current techniques and research in this area see Ottova and Tien, 1997.
Phospholipase C (PLC) is a generic name for enzymes that catalyze the hydrolysis of phosphoglycerides into diacylglycerols and phosphorylated alcohols such as serine, choline, inositol, glycerol, or ethanolamine. For example, a specific phospholipase C hydrolyzes phosphatidylinositol-4-phosphate (PIP) or phosphatidylinositol-4,5-bisphosphate (PIP.sub.2), resulting in each case in the formation of two second messengers: a hydrophobic diacylglycerol and a hydrophilic inositol phosphate (IP.sub.2 or IP.sub.3 respectively). This hydrolysis can be monitored by a variety of methods using endogenously or exogenously labeled substrates.
De Vivo (1994) describes current methods used for measuring the hydrolysis of PIP.sub.2. In the endogenous substrate approach, the cells of interest are cultured in the presence of myo-[.sup.3 H]-inositol. The cells convert inositol to phosphatidylinositol (PI) using phosphatidylinositol synthase, and the PI in turn is converted to PIP-[.sup.3 H] and PIP.sub.2 -[.sup.3 H] by phosphatidylinositol kinases. When the polyphosphoinositide pool is labeled to a steady state, the breakdown of PIP and PIP.sub.2 is initiated by the addition to the cell culture medium of an appropriate stimulatory factor (e.g., receptor agonists for intact cells or guanylyl nucleotides for permeabilized cells).
In the exogenous substrate approach, purified labeled phospholipids are used as the substrate. The phospholipids are mixed in the presence or absence of detergent and sonicated briefly on ice to prepare vesicles. Aliquots of the substrate are mixed with a source of PLC (membranes or purified enzyme) and, often, G-protein subunits.
In both the endogenous and exogenous substrate approaches, solvent extraction is used to separate the hydrophilic reaction products from the hydrophobic substrate. Current methods for sphingomyelinase assays also require solvent extraction steps. It would be desirable to eliminate the extraction step for environmental and health reasons. In addition, it is difficult to automate the extraction of large numbers of samples, as would be necessary for example in high throughput screening of drug candidates.